Despite numerous positive animal studies, clinical neuroprotection trials after traumatic brain injury (TBI) have uniformly failed with regard to primary end points and general populations. However, most such therapies have been directed toward a single proposed injury pathway and have targeted very early biochemical changes. Our prior work has shown that cell cycle activation is a key component of secondary injury following TBI; based upon our research and that from other groups, we hypothesize that E2F 1, 2, and 3 are involved in both cell cycle-related neuronal death (CRND) and microglial activation after brain trauma. In the present project we propose to use highly specific E2F decoy oligonucleotides (ODN) to demonstrate the neuroprotective effect of blocking the E2Fs. We will generate cell specific and inducible E2F 1, 2, and 3 knockouts and Prohibitin-1 knock-in to demonstrate that cell cycle activation in neurons and microglia represent separate and additive secondary injury mechanisms. We will also use the retinoblastoma modulator RRD-251 to confirm the neuroprotective effect of blocking the E2Fs. Moreover, we propose to expand our focus beyond the acute injury and test the hypothesis that delayed cell cycle activation contributes to chronic neurodegeneration and microglial activation months after TBI. Specific aims are to show that: (1) TBI-induced activation of E2F transcription factors is a critical event contributing to acute neuronal cell death and microglial/astrocyte reactivity, and that treatment with highly specific E2F decoy oligonucleotide (ODN) sequences or the Rb modulator RRD-251 attenuates these cellular changes and limits injury-induced pathobiology (2) E2F 1-3 are important contributing initiators of neuronal cell death and activation of microglia, with attenuation of E2F activity in neurons, and microglia showing additive neuroprotective effects; conditional and cell specific triple knockouts (E2F 1-3) will be generated by mating E2F 1-3 loxP mice with our inducible neuronal and microglia specific Cre mice; conditional and cell specific Prohibitin-1 knock-in will be generated by mating Prohibitin-1 loxP mice with our inducible neuronal and microglia specific Cre mice (3) Cell cycle activation after TBI persists beyond the acute period and leads to progressively increased polyploidy of certain neurons in selectively vulnerable brain regions as demonstrated by slide-based cytometry and fluorescence in- situ hybridization (FISH); these changes are not followed by rapid neuronal death but rather serve to predispose to chronic progressive neurodegeneration (4) Induction of conditional, neuron specific or microglial specific triple knockouts (E2F 1-3), or treatment with a the pan-CDK inhibitor CR8, at 1 month after trauma, reduce progressive neuronal hyperploidy, and attenuate chronic progressive neuronal loss and associated neurological impairment detected at 1 year after trauma.